Year: 1999
Type: article
Abstract: A question of central importance to the molecular endocrinologist is how hormones function to orchestrate events within cells. The cascades of cellular responses that are triggered by endocrine signals require the formation of specific protein partnerships, and these protein-protein interactions must be coordinated in both space and time. For example, in the absence of ligand, the steroid hormone receptor for estradiol is associated with a multiprotein inhibitory complex (1). The binding of estradiol results in alterations in estrogen receptor conformation that allow it to dissociate from this complex, and the receptor becomes competent to interact with specific DNA elements in the regulatory regions of target genes. The efficient utilization of these regulatory elements by the receptor, however, requires that the receptor associate with other coregulatory proteins (2–4). Biochemical approaches such as coimmunoprecipitation and Far-Western blotting and in vivo approaches such as yeast two-hybrid assay have provided important information regarding the interactions between receptors and coregulatory proteins. These approaches, however, may sometimes implicate nonphysiological associations between proteins that do not normally occur in intact cells. Deciphering where and when specific protein partnerships form within the living cell will be critical to understanding these basic cellular events. The molecular cloning of the jellyfish green fluorescent protein (GFP) and its expression in a variety of cell types have had a major impact on our ability to monitor events within living cells (5–10). GFP retains its fluorescent properties when fused to other proteins, and this allows fluorescence microscopy to be used to monitor the dynamic behavior of the expressed GFP fusion proteins in their natural environment within the living cell. There are now many examples of proteins expressed as GFP chimeras that possess the same subcellular localization and biological function as their endogenous counterparts. For example, the dynamics of nuclear translocation for the glucocorticoid (11, 12) and androgen receptors (13) have been visualized using GFP fusions. GFP fusion proteins have also been used to monitor complex cellular events such as the sorting of proteins between organelles (14) and the dynamics of regulated protein secretion (15, 16). Recently, it was demonstrated that movement ofβ -arrestin2-GFP fusion proteins serves as a sensitive biosensor for G protein-coupled receptor activation (17). This illustrates the potential for GFP fusion proteins to act as indicators of many different intracellular events. Mutant forms of the GFP protein that emit lights of different colors have been generated that, when coexpressed in the same cell, can be readily distinguished by fluorescence microscopy. This allows the behavior of two independent proteins to be monitored in the intact cell, and the extent to which these proteins colocalize can be assessed. To determine whether these labeled proteins are physically interacting, however, would require resolution beyond the optical limit of the light microscope. Fortunately, this degree of spatial resolution can be achieved with the conventional light microscope using the technique of fluorescence resonance energy transfer (FRET). FRET microscopy involves the detection of increased (sensitized) emission from an acceptor fluorophore that occurs as the result of the direct transfer of excitation energy from an appropriately positioned fluorescent donor. The efficiency of energy transfer from the donor to the acceptor is extremely sensitive to the distance between the fluorophores and is limited to the scale of nanometers (18). As will be discussed below, the spectral characteristics of some of the mutant variants of GFP allow them to be used as donor and acceptor pairs for FRET microscopy. The expression of nuclear proteins fused to color variants of GFP provides a method to visualize where and when two independent nuclear proteins localize within the nuclear compartment of intact cells. When combined with FRET microscopy, this approach has the potential to report dynamic changes in protein-protein interactions as they occur in the nucleus. These types of studies will complement the biochemical and two-hybrid experimental approaches and will have important implications for understanding the mechanisms underlying gene regulation. In this paper, we review the characteristics of the GFPs that make them useful for the study of nuclear protein behavior in living cells. The jellyfish GFP is a 27-kDa protein that absorbs near-UV light and emits green light. GFP owes its fluorescent properties to a chromophore that forms by a posttranslational oxidation and cyclization reaction involving the tripeptide sequence, Ser65, Tyr66, Gly67 (19–22). The crystal structure of GFP reveals an 11-stranded β-barrel cylinder surrounding the central chromophore (23). Because the protein must fold into this barrel structure for appropriate formation of the chromophore, nearly the entire 238-amino acid sequence is necessary for fluorescence (24). In addition, the formation of the chromophore is a relatively slow process requiring several hours (25, 26). Therefore, it is critical that protein fusions to GFP do not interfere with the ability of the chromophore to form and, when imaging newly synthesized GFP fusion proteins, it is important to consider the rate of chromophore formation. The major limitation to the detection of GFP in living cells is the autofluorescent background. For cells grown as a monolayer in culture, this background is primarily due to intracellular NAD(P)H, riboflavin, flavin coenzymes, and flavoproteins bound in the mitochondria (27). In more complex cell cultures, such as slice preparations from transgenic animal tissues, certain specialized structures may contribute significantly to this fluorescent background (28). The autofluorescent signal can be substantial at near-UV wavelengths, and this has limited the detection of wild-type GFP (wtGFP) to approximately 105 molecules per cell (7). Fortunately, mutagenesis of the wtGFP protein sequence has generated variant forms with increased brightness and differing spectral characteristics (19, 23, 25, 29). A mutation changing the chromophore Ser65 to threonine (GFPS65T) resulted in a shift in excitation away from the near-UV to a peak at 489 nm and yielded a 4- to 6-fold improvement in the intensity of green light emission when compared with wtGFP (25). Improvements in the expression of GFPS65T in mammalian cells was obtained by optimizing codon usage to facilitate its translation (30–32) and by the introduction of mutations that enhance protein folding at 37 C (33, 34). Taken together, these mutations have dramatically improved the fluorescence signal obtained from GFP chimeras, allowing the detection of fewer than 10,000 GFP molecules in single living cells (26). Mutations within the chromophore have also produced several color variants of GFP. For example, changing Tyr66 to His resulted in a blue fluorescent protein (BFP) that, when excited by UV light, has a peak emission at 445 nm (19, 29). BFP is more difficult to detect than the GFP variants, however, because of its low quantum yield and sensitivity to photobleaching. Other GFP variants with emission wavelengths from cyan (greenish blue) to yellowish green have been isolated (23, 29), and these may prove more useful than BFP and GFPS65T for some applications. However, because the peak emission values for BFP and GFPS65T are substantially different (445 nm and 511 nm, respectively), BFP has utility when used in conjunction with GFPS65T as a second protein tag (Refs. 35, 36 ; see below). Plasmid vectors encoding several of the color variants of GFP that have been optimized for expression in mammalian cells are commercially available. These vectors allow for fusion of GFP to any cloned gene of interest, and standard gene transfer techniques are used to introduce these vectors into cells for the expression of the chimeric proteins. The detection of GFP fusion proteins does not require the addition of substrates or the permeabilization and fixation of cells. This improves the sensitivity and reduces the potential for artifacts associated with immunohistochemical approaches (37). The primary consideration in the generation of GFP fusion proteins is functionality, both of the fluorescent protein tag and the protein of interest. As mentioned above, the GFPs must fold correctly when positioned at either the amino- or at the carboxy-terminal end of the protein of interest. Equally important is that the protein being studied retains its normal cellular function when fused to the 27-kDa fluorescent protein. Therefore, it is critical that the expressed fusion proteins be tested for function by as many independent methods as possible. For example, fluorescence microscopy of transiently transfected cells will show that the GFP chimeras are being expressed, reveal their subcellular localization, and indicate the level of the GFP signal over the autofluorescence background. Confirmation that the expressed fusion proteins are full length is then obtained by Western blotting of proteins extracted from the transfected cells. Antisera directed against the protein of interest and against GFP can be used sequentially to identify the expressed protein. Antibodies for detection of GFP that are suitable for Western analysis are commercially available. Further, it is important to identify a method that directly demonstrates that protein function is not impaired by the GFPs. For example, GFP fusions to transcription factors can be tested for binding to appropriate DNA elements using electrophoretic mobility shift assay, and their ability to influence specific gene transcription can be determined in reporter gene studies (38). A more rigorous demonstration of GFP chimera function is the capacity of the fusion protein to rescue a mutant phenotype in a transgenic organism. For example, Take-Uchi and colleagues (39) recently showed that expression of a GFP-sodium channel fusion protein in a mutant strain of Caenorhabditis elegans could reestablish a complex rhythmic behavior. The coexpression of BFP- and GFPS65T fusion proteins, combined with dual channel fluorescence microscopy, provides a method for monitoring the intracellular trafficking of two independent proteins in the same cell (35, 36). When GFP chimeras are imaged in intact cells, there are several important considerations that become especially relevant when using dual-channel fluorescence microscopy. The choice of the basic components of a fluorescence microscope the excitation and has important for For example, a light at certain wavelengths, and its blue of emission are for excitation of GFP. However, a with its from the UV to may be for excitation of BFP and for studies involving the imaging of two different color efficient excitation at requires the of that are to UV light. is also important to that in the can when fluorescence signals at two different wavelengths are to the with different This is for in imaging that of the and The efficiency of light the brightness of the fluorescence and the ability to are both of the of the The of a will the of light to the and resolution and However, resolution can be to of the that occurs when light from the of the is than that from the are for this requires a in the light Therefore, a that for in will be than for imaging living cells in Recently, have become that the and resolution of obtained from living cell are to the blue light emission of BFP from the green light emission of Because the cellular autofluorescence signal has a emission will the of fluorescence signals the will also the due to the of the excitation and emission for the BFP and GFPS65T variants it is important to an excitation for BFP that has a excitation of that are for the detection of the different color variants of GFP are now commercially available. the low quantum yield of BFP and its to for its detection the background Because BFP and GFPS65T at different the using dual channel will the of the cell. is important to a with sensitivity at blue and green wavelengths to the intensity and of to the excitation light that is for detection of BFP fusion proteins. Because of their sensitivity and are used in conventional fluorescence microscopy. The with the sensitivity are to and that light directly to the is that transcription factors are to specific within the The steroid hormone for example, to be regulated in their of fluorescence microscopy of cells nuclear proteins fused to the BFP- and GFPS65T spectral variants allows the of the of for two different proteins within the of living cells. we this approach by and the localization of the transcription the estrogen receptor and the glucocorticoid protein is the of the nuclear receptor that directly with the of several different nuclear receptors The results in how signals from cells pairs of these nuclear proteins with the GFP or BFP variants can be by dual-channel fluorescence microscopy. In this approach is used to of a cell and The for fluorophore are to using the green channel to indicate GFP and the blue channel to indicate The GFP and BFP from the same cell are then into a single to the in the localization of the labeled proteins. For this the GFP is to the green the BFP is now to the channel of the same In this signals from proteins as in the signals green and In the in for a cell and the reveals of by in as as regions where and are in their localization by in more of different localization is for cells and The of in the of the cell in is from the for the coexpressed protein In when and are coexpressed in cells, we of nuclear The of the labeled estrogen receptor and proteins to the same could function to facilitate interactions between these proteins in for a between and the estrogen receptor from the that and interactions in by coimmunoprecipitation studies is however, is a direct demonstration of interactions between these proteins in the living cell. The studies are limited by the optical resolution of the light microscope and can indicate on the scale of approximately To determine whether these labeled proteins are physically requires resolution beyond the optical limit of the light microscope. This degree of spatial resolution can be achieved by conventional light microscopy using the technique of FRET microscopy. of the for and cells transfected with expression vectors encoding the fusion proteins by and used to on The in with endogenous at C and then to 37 The fluorescence with a using a excitation light The nm excitation and nm emission for GFP nm excitation and nm emission for BFP of the cells obtained using a with of with The to GFP to the green channel and BFP to the blue The then by the GFP to the green channel and the BFP to the channel of the same of the of a cell scale and the is the by the in the The GFP and BFP as to show regions of by and regions where and are in their localization by or green in of the of a cell and The GFP and BFP the nuclear for these two fusion proteins. of the from two cells the and fusion proteins. The a degree of in the for these two fusion proteins by FRET microscopy the fluorescence emission from acceptor fluorophores that results from the direct transfer of excitation energy from appropriately positioned donor This requires that there be a substantial in the emission of the donor with the of and some of the GFP color variants have the spectral For BFP can excitation energy to GFPS65T and the cyan color variant can as a donor for the yellowish mutant Because the efficiency of energy transfer with the of the distance the donor and acceptor FRET can occur over a distance limited to approximately To this in the a complex of proteins and is approximately and the light microscope could in of or In the detection of fluorescence emission by FRET microscopy reveals that the distance proteins labeled with the color variants of GFP is on the of the of a single protein that is of the the of the spectral variants of the of FRET to living cells was limited to fluorescent directed to the cell or to into cells FRET microscopy can detect interactions between any proteins that biological function when expressed as a fusion to the GFPs. approaches that of the of the GFPs and FRET imaging have recently been used to monitor intracellular events. The approach involves the detection of FRET signals that from chimeric proteins donor or the cyan and acceptor or the yellowish fluorophores a that the binding for The of the with the a in the of the fluorophores and the FRET FRET signals from chimeric proteins a that or has to be a sensitive of in living cells the ability to target the expression of these fusion proteins to specific cellular organelles allows for the monitoring of changes in in the intact cell These studies the utility of FRET as a reporter of dynamic intracellular events and the of that will indicate a variety of in intact cells A second FRET approach involves the detection of interactions between two different protein labeled with the GFPs In this the labeled proteins are not limited in the distance they can be the detection of fluorescence emission from pairs of proteins labeled with two different color variants of GFP can direct for interactions between these proteins. This approach has the potential of being more than FRET in that it can be used to the interactions between many different of cellular proteins. of and FRET between to the of GFP how FRET between two color variants of GFP fused to by a can as a biosensor for the binding of a In the absence of the the GFPs beyond the critical distance for energy transfer binding of ligand, a in conformation of the can the GFPs within the necessary for in green light emission when BFP is excited how FRET between two independent proteins labeled with the GFPs can indicate protein-protein For proteins of the cells to BFP results in emission of blue light In of the cells fusion proteins can result in emission of green light because of FRET from BFP to an that the proteins are in However, a result using the FRET technique does not that the proteins to interact as in The results in the of FRET signals from living cells using both the and In the example, FRET is from cells a chimeric protein in which GFP is to BFP a acid The cells the fusion protein by green and acceptor then by BFP and blue light emission and then by BFP and green light emission using the in the of The donor and acceptor are combined into a single and a is to indicate the fluorescence signal intensity To facilitate the direct of signal the level intensity for both donor and acceptor fluorescence the of the two cells in was determined and the results are of cells the protein show that the acceptor signal was than the donor signal These results are with the transfer of BFP excitation energy to the GFP the emission of green light. FRET of GFP and BFP The same GFP and BFP used as in the of the acceptor is nm excitation and nm FRET obtained using a slow with a imaging The of the is with donor and acceptor using a time. fluorescence signals within the of to level intensity background and of the had The was used to the and determine the level intensity of two cells the fusion protein obtained using the GFP BFP and the acceptor The donor and acceptor are combined in a single and a was to facilitate of fluorescence signal intensity signal level with being A was cell and the level intensity for this was for both the donor and acceptor The donor and acceptor signal level intensity determined for cells was then of cells the and proteins obtained using the GFP BFP and the acceptor and a of donor and acceptor fluorescence was as The level intensity of the cell was and these are for both the donor and acceptor The donor and acceptor level intensity for the cells was determined and cells the and proteins in with endogenous obtained using the GFP BFP and the acceptor The of donor and acceptor fluorescence was and the level intensity the two cell is for both the donor and acceptor The in C are from with the of this FRET imaging approach to visualize the of proteins labeled with GFP and BFP in the of living cells (38). we the coexpression of these protein to the of FRET cells the fusion proteins by green fluorescence and acceptor of cells then using the same for the protein. A of the background donor and acceptor was obtained and the same was to indicate fluorescence intensity level intensity of the in and the results the acceptor signals the donor signals by that energy transfer from to requiring that these proteins be in A limitation to the FRET imaging approach is that the to detect green light emission from a of labeled proteins can not be as an that these proteins do not physically For example, we have the FRET imaging approach to monitor the formation of partnerships between and other nuclear proteins, the estrogen receptor (38). interactions between and the estrogen receptor are a in the of gene and a of these two proteins was demonstrated by in techniques The results in show FRET imaging of cells the estrogen receptor labeled with GFP and the fusion protein. The the donor and acceptor reveals that the fluorescence intensity for the acceptor is significantly than that of the donor The level intensity the two in that the acceptor signal is approximately than the donor These results are to obtained for proteins labeled with the GFPs (38). There are many potential protein may to FRET transfer is not the distance the their as In the of proteins labeled with the the fluorophores are positioned at the of the proteins. The that are by the proteins when they associate may not allow the fluorophores to be in or to appropriately for energy transfer to occur Further, the endogenous of the labeled proteins will also interact with the expressed GFP chimeras, for potential This can be by the labeled proteins in cell types that the endogenous proteins, or by the labeled protein in of the endogenous proteins. However, level expression of proteins that are within the cell, not directly interacting, could allow FRET to occur by As with any approach involving the expression of proteins in living cells, artifacts that from of the fusion proteins are a with labeled proteins that that not physically can be used to the of to FRET A limitation of FRET microscopy, especially when with two independent proteins in the of living cells, is the of for spectral for both the donor and acceptor This occurs when the donor emission into the acceptor and when the acceptor emission occurs at the donor excitation The result of spectral is a and background against which FRET signals must be The detection of FRET using the GFPs is also limited by the of the of the expressed donor and acceptor fusion proteins. Therefore, to a for is necessary to Recently, a method for FRET efficiency was This method the sensitivity of FRET by for the for donor and acceptor when they substantial spectral This approach was used by and colleagues to protein-protein interactions between two proteins in the of and fluorescence microscopy that the and proteins are to the and FRET demonstrated that these proteins yeast two-hybrid assay is the method of choice for protein However, not protein partnerships by yeast two-hybrid The that these protein interact in a in vivo can be difficult to FRET provides a to these protein-protein interactions in the intact cell. The potential for FRET microscopy of living cells labeled fusion proteins has to be approaches are being that different energy transfer for GFP that may significantly the detection of protein For example, and colleagues have a fluorescent that can cell and to proteins that are with a the The is becomes becomes fluorescent when it to the excitation and emission make it suitable as an acceptor of excitation energy from the cyan mutant of GFP. The ability to the acceptor fluorophore to cells the donor provides a useful for in that the donor signal can be in the and then in the of the acceptor fluorophore A second approach of the energy transfer used by the jellyfish In the the emits blue light that green light emission from GFP. by have demonstrated that resonance energy transfer between the and yellowish color variant of to as resonance energy transfer may have several over the FRET Because the donor is a spectral and autofluorescence are not a in using the The have the approach to the formation of involving the protein in These in the combined of proteins and FRET microscopy. For many important such as the of transcription by the nuclear receptors and their coregulatory the critical events are coordinated in space and interactions within complex The of the FRET approach is in the detection of these between regulatory proteins in their natural environment within the living cell. of the information in this is the result of with our colleagues and The to of the for at the of for are to for the encoding and the of This study was by for and
Source: Molecular Endocrinology
DOI: 10.1210/me.13.4.517
Authors: Richard N. Day, Steven K. Nordeen, Yihong Wan
Institutions: University of Virginia, University of Colorado Denver
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2042337136